Preparation of potyvirus VPg

VPg from turnip mosaic virus was expressed and purified using the established protocol described elsewhere [25]. The cDNA clone of TuMV VPg plasmid was constructed from the pET-21a and the resulting construct was subjected to digestion with NdeI and XhoI restriction enzymes. His-tagged VPg was expressed [25] in BL21 (DE3) pLysS E. coli cells. The growth of cells took place in Luria-Bertani (LB) medium at a temperature of 37°C. Following induction with 100 μM isopropyl 1-thio-β-D-galactopyranoside (IPTG) for 6 hours at 37°C, the cells were harvested, the cell pellets were resuspended in a 20 mM Tris-HCl, pH 7.5 buffer containing 100 mM NaCl at 4°C, and cell disruption was achieved through sonication. The supernatant was passed through a Ni-Sepharose column (Novagen), and the protein bound to the column was eluted with a 20 mM Tris-HCl, pH 8.0 buffer containing 100 mM NaCl and 250 mM imidazole. The sample was subjected to dialysis using a 20 mM Tris-HCl, pH 8.0 buffer supplemented with 1 mM DTT and 5% glycerol. The protein sample was subsequently loaded onto a HiTrap-Mono-Q anion exchange column. The sample was subjected to dialysis against a 20 mM Tris-HCl, pH 7.6 buffer supplemented with 50 mM NaCl, and 5% glycerol. The purity of the VPg protein was subsequently verified through 15% SDS-PAGE analysis. All samples were dialyzed against a 20 mM Tris-HCl, pH 7.6 buffer containing 100 mM NaCl, 1 mM MgCl₂, 1 mM DTT, and 0.1 mM EDTA, and subsequently subjected to filtration through a 0.22 μM Millipore filter prior to the spectroscopy measurements. The protein concentrations were quantified using a Bio-Rad protein assay reagent (Bio-Rad Laboratories, CA) and the Bradford assay, employing bovine serum albumin as the standard [35].